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Promotion of Growth and Physiological Characteristics inWater-Stressed Triticum aestivum in Relation toFoliar-Application of Salicylic Acid
Abida Parveen 1,*, Muhammad Arslan Ashraf 1, Iqbal Hussain 1 , Shagufta Perveen 1, Rizwan Rasheed 1 ,Qaisar Mahmood 2,*, Shahid Hussain 1, Allah Ditta 3,4 , Abeer Hashem 5,6, Al-Bandari Fahad Al-Arjani 5,Abdulaziz A. Alqarawi 7 and Elsayed Fathi Abd Allah 7
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Citation: Parveen, A.; Arslan Ashraf,
M.; Hussain, I.; Perveen, S.; Rasheed,
R.; Mahmood, Q.; Hussain, S.; Ditta,
A.; Hashem, A.; Al-Arjani, A.-B.F.;
et al. Promotion of Growth and
Physiological Characteristics in
Water-Stressed Triticum aestivum in
Relation to Foliar-Application of
Salicylic Acid. Water 2021, 13, 1316.
https://doi.org/10.3390/w13091316
Academic Editor: Teresa Afonso
do Paço
Received: 28 February 2021
Accepted: 8 April 2021
Published: 8 May 2021
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1 Department of Botany, Government College University, Faisalabad 38000, Pakistan;[email protected] (M.A.A.); [email protected] (I.H.); [email protected] (S.P.);[email protected] (R.R.); [email protected] (S.H.)
2 Department of Environmental Sciences, COMSATS University Islamabad, Abbottabad Campus,Peshawar 24730, Pakistan
3 Department of Environmental Sciences, Shaheed Benazir Bhutto University, Sheringal, Upper Dir,Khyber Pakhtunkhwa, Peshawar 18050, Pakistan; [email protected]
4 School of Biological Sciences, The University of Western Australia, 35 Stirling Highway,Perth, WA 6009, Australia
5 Botany and Microbiology Department, College of Science, King Saud University, P.O. Box 2460,Riyadh 11451, Saudi Arabia; [email protected] (A.H.); [email protected] (A.-B.F.A.-A.)
6 Mycology and Plant Disease Survey Department, Plant Pathology Research Institute, ARC, Giza 12511, Egypt7 Plant Production Department, College of Food and Agricultural Sciences, King Saud University,
P.O. Box 2460, Riyadh 11451, Saudi Arabia; [email protected] (A.A.A.); [email protected] (E.F.A.A.)* Correspondence: [email protected] (A.P.); [email protected] (Q.M.)
Abstract: The present work reports the assessment of the effectiveness of a foliar-spray of salicylicacid (SA) on growth attributes, biochemical characteristics, antioxidant activities and osmolytesaccumulation in wheat grown under control (100% field capacity) and water stressed (60% fieldcapacity) conditions. The total available water (TAW), calculated for a rooting depth of 1.65 mwas 8.45 inches and readily available water (RAW), considering a depletion factor of 0.55, was4.65 inches. The water contents corresponding to 100 and 60% field capacity were 5.70 and 1.66 inches,respectively. For this purpose, seeds of two wheat cultivars (Fsd-2008 and S-24) were grown in potssubjected to water stress. Water stress at 60% field capacity markedly reduced the growth attributes,photosynthetic pigments, total soluble proteins (TSP) and total phenolic contents (TPC) comparedwith control. However, cv. Fsd-2008 was recorded as strongly drought-tolerant and performed bettercompared to cv. S-24, which was moderately drought tolerant. However, water stress enhanced thecontents of malondialdehyde (MDA), hydrogen peroxide (H2O2) and membrane electrolyte leakage(EL) and modulated the activities of antioxidant enzymes (superoxide dismutase (SOD), peroxidase(POD), and catalase (CAT), as well as accumulation of ascorbic acid (AsA), proline (Pro) and glycinebetaine (GB) contents. Foliar-spray with salicylic acid (SA; 0, 3 mM and 6 mM) effectively mitigatedthe adverse effects of water stress on both cultivars. SA application at 6 mM enhanced the shootand root length, as well as their fresh and dry weights, and improved photosynthetic pigments. SAfoliage application further enhanced the activities of antioxidant enzymes (SOD, POD, and CAT) andnonenzymatic antioxidants such as ascorbic acid and phenolics contents. However, foliar-spray ofSA reduced MDA, H2O2 and membrane permeability in both cultivars under stress conditions. Theresults of the present study suggest that foliar-spray of salicylic acid was effective in increasing thetolerance of wheat plants under drought stress in terms of growth attributes, antioxidant defensemechanisms, accumulation of osmolytes, and by reducing membrane lipid peroxidation.
Keywords: drought stress; osmolytes; salicylic acid; wheat; proline; photosynthetic pigments
Water 2021, 13, 1316. https://doi.org/10.3390/w13091316 https://www.mdpi.com/journal/water
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1. Introduction
The distribution of plant-life is largely dependent on water in comparison to with otherabiotic factors [1]. Important crops show a considerable decline in growth and productivitydue to the limited availability of water, which provides a challenge to food security world-wide. In this context, plant crop plants show sensitivity to drought stress at early stagesthan at other developmental stages [2]. Greater intensity of drought stress severely reducedgrowth attributes and biomass in maize [3,4]. Drought severely affects key physiologicaland biochemical processes in crop plants such as photosynthetic rate, stomatal regulation,cell growth, water relations, nutrient metabolism and hormonal regulation [1,5,6]. Scarcityof water affects morphological, physiological and biochemical mechanisms, eventuallyreducing productivity [3,7–9]. Water stress induces overproduction of reactive oxygenspecies (ROS), such as singlet oxygen species (1O2), hydrogen peroxide (H2O2), superoxideanions (O2-) and hydroxyl radical (OH-) which damage membrane lipids, cellular proteins,chlorophyll pigments and nucleic acids [10,11]. Under severe stress, this damage leads tointracellular death and eventually plant death [12]. There is considerable interest in ROSproduction within organelles such as chloroplasts and vacuoles, as well as in microbesand mitochondria [13]. Plants adopt protective mechanisms, which can quickly detoxifyROS produced due to drought stress [14,15]. From the defense perspective, enzymaticantioxidants neutralize or protect against oxidative damage. Furthermore, superoxidedismutase (SOD) plays a considerable role, detoxifying many cellular organelles, whileperoxidase (POD) and catalase (CAT) neutralize potential oxidants, which leads to plantresistance under osmotic stress [13]. If a plant under stressful conditions experiences adecline in antioxidant levels inhibition of photosynthetic machinery and loss of cell func-tions may occur [16]. Among nonenzymatic antioxidants such as carotenoids, ascorbic acid(AsA) and phenolics play an important role in defensive mechanisms, as well as in themaintenance of key cellular characteristics [17,18]. However, it has been documented byseveral researchers that antioxidants mitigate osmotic damage and confer stress tolerancein crop species such as maize [19], radish [20] and wheat [21].
To overcome the damaging impacts of drought stress, crop plants develop defensivemechanisms such as the production of osmolyte-assisted proline (PRO), glycine betaine(GB), total soluble proteins (TSP), secondary metabolites and carbohydrates [22–24]. It wasfound that osmotic adjustment reduced osmotic potential and displayed beneficiaries interms of increased growth and moderate cell turgor levels, as well as improving metabolicfunctions of the cell [25]. Similarly, it was observed that glycine betaine accumulated inmaize under water deficit conditions, which led to enhanced growth characteristics [26].In addition to this, proline in cotton plants under water-stressed conditions serves as anefficient osmolyte during osmoregulation [14]. However, plants show variation in responseto tolerance of various abiotic stresses, and various strategies have been developed byplants to tolerate abiotic stresses for better growth and yield [6,27,28]. It was foundby [29] in Brassica napus that plants under drought-stressed conditions developed stresstolerance mechanisms.
Salicylic acid (SA) is a water-soluble, phenolic compound and an endogenous growthregulator, which participates in the regulation of physiological processes in plants as asignaling molecule. Foliar-spray of SA improved growth characteristics in plants, includingshoot length, biomass, chlorophyll pigments and carbohydrate content, as well as yieldcharacteristics [30]. Similarly, SA is involved in activation of physiological processes inplants such as stomatal regulation, nutrient uptake, chlorophyll synthesis, protein synthesis,inhibition of ethylene biosynthesis, transpiration and photosynthesis [31,32]. As a growthregulator, SA is involved in mechanisms to cure diseases after pathogen attack [33]. Inaddition to this, salicylic acid exerts a growth stimulative impact by direct interferingwith enzyme activities by activating growth-promoting cellular activities. SA plays a keyrole in mitigating numerous abiotic stresses, such as drought tolerance in wheat [34] andcadmium tolerance in barley [35]. Furthermore, SA induces resistance of seedlings toosmotic stress [36], chilling and high temperature by increasing the activities of glutathione
Water 2021, 13, 1316 3 of 17
reductase, as well as increasing guaiacol peroxidase [36] activity against the toxicity ofheavy metals [37].
Wheat (Triticum aestivum L.) is a major cereal crop, a major source of calories andprotein, and is cultivated at a global level. It is estimated that the wheat requirementof humans will increase by up to 60% by 2050 [37]. However, its production is severelyhampered by various abiotic stresses, including drought stress [38]. The current inves-tigation indicates that salicylic acid, as an endogenous growth regulator, reduces theadverse effects of drought-induced osmotic stress by modulating antioxidant defense,and physio-biochemical mechanisms. No reports, or very few, are available about thefoliar application of salicylic acid, and it being a signaling molecule in terms of activatingantioxidant enzymes. This study considered whether or not foliar spray of SA could bringfavorable physiological changes to enhance growth in drought-stressed wheat. Thus, theprincipal objective of the study was to assess whether foliar-applied salicylic acid couldalter antioxidant capacity, osmolyte accumulation and photosynthetic pigments to enhancethe growth of wheat seedlings exposed to drought stress.
2. Materials and Methods
The present study was conducted to investigate the effect of varying water-limitedregimes on different wheat cultivars. Experiments were done in plastic pots placed inthe greenhouse of the Botanical Garden of Government College University, Faisalabad,Pakistan to study the effect of foliar-application of a plant growth regulator (SA) ontwo drought-tolerant wheat cultivars (Fsd-2008, and S-24). A boom sprayer was usedwith a hollow cone nozzle capable of spraying at a pressure of 50 psi. Fsd-2008 is astrongly drought-tolerant wheat cultivar, and S-24 is a moderately drought-tolerant wheatcultivar, based on various morphological, physiological, and biochemical parameters.Seeds of wheat cultivars were obtained from Ayub Agricultural Research Institutes (AARI),Faisalabad, Pakistan. After surface sterilization of seeds with 0.1% HgCl2 for 2 min seedswere washed with deionized water. Ten seeds were sown per replicate in each plastic pot,filled with 25–30 cm with 10 kg sandy clay loam soil. The design of the experiment was acompletely randomized design with three replicates per treatment. Later on, after seedlingemergence, five seedlings were maintained in each pot after seven days of germination. Theexperiment consisted of two sets of pots. One set was supplied with normal irrigation (100%field capacity) and another set was drought-stressed (60% field capacity). Soil moisturelevel was calculated on a soil dry-weight basis. To check evapotranspiration, pots wereweighed at two day-intervals so the soil moisture level inside the pot was kept at 100%and 60% field capacity, precisely, according to treatments, which maintained withholdingwatering. The total available water (TAW), calculated for a rooting depth of 1.65 m was8.45 inches, and readily available water (RAW), considering a depletion factor of 0.55,was 4.65 inches. The water contents corresponding to 100 and 60% field capacity were5.70 and 1.66 inches, respectively. Fertilizer requirements (i.e., N, P, and K) were fulfilledusing urea, diammonium phosphate (DAP) and sulfate of potash at 0.03 and 0.025 g/kg ofsoil, respectively. Soil pH was between 6.6–7.2. Daily climatic conditions on an averagebasis were recorded showing a day/night length of 14/10 h, minimum and maximumday/night temperatures of 18–9 ◦C and 25–15 ◦C, respectively, and a daytime averagerelative humidity of 50%. Drought stress at 60% field capacity was imposed to seedlingsafter one week of germination, then seedlings were allowed to grow for two more weeksunder varying water regimes. After two weeks of water deficit treatment, the seedlingswere subjected to foliarly-applied levels of salicylic acid at 3 mM or 6 mM. The solution of(500 mL) plant growth regulator was prepared in distilled water containing 0.1% Tween-20. After one week of foliar application, data were recorded for various attributes of theseedlings in terms of shoot and root length, and fresh weights were recorded at harvesttime. For measuring dry weights, shoots and roots were kept at 70 ◦C for three weeks.For the estimation of biochemical attributes, fresh leaves were collected and preserved at−20 ◦C before harvesting.
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Photosynthetic attributes. The fresh leaf photosynthetic pigments chlorophyll a (Chla), chlorophyll b (Chl b) and carotenoids were estimated according to the method of [39],using acetone as an extract. Fresh leaf material for the sample extract (0.1 g) was put into10 mL of an 80% acetone containing overnight at 4 ◦C. The absorbance of the extract wasmeasured on 663 nm, 645 nm and 480 nm (Hitachi U-2001, Tokyo, Japan)
Proline estimation. Proline was estimated by applying the protocol of [40]. Sampleswere homogenized using sulfosalicylic acid (3% w/v), and after filtration free prolinecontents were estimated.
Glycine betaine estimation. The method of [41] was employed for the determinationof GB content in fresh leaf samples. Leaf samples were ground in 0.5% toluene (10 mL) andthe mixture was filtered.
Malondialdehyde (MDA). MDA contents were determined by applying the methodof [42]. Wheat leaves (1.0 g) were extracted in 0.1% TCA solution, and the mixturecentrifuged for 15 min. Afterwards, 0.5 mL of supernatant was mixed with 3 mL of0.5% thiobarbituric acid (TBA) solution prepared in 20% TCA. After shaking the mix-ture, the MDA content of the supernatant was determined at 532 nm and 600 nm usinga spectrophotometer.
Hydrogen peroxide (H2O2). Trichloroacetic acid (0.1% w/v) was used for the estima-tion of H2O2 employing the protocol of [43]. The homogenized material was centrifuged at15,000 rpm. The supernatant was mixed with 1 mL of KI solution and absorbance measuredat 390 nm.
Relative membrane permeability (RMP). The method of [44] was used to determinethe membrane stability characteristics of fresh leaves.
Total phenolics. The method of [45] was used to estimate total phenolic content infresh leaves. Fresh wheat leaves (0.5 g) were extracted in 80% acetone and the extractcentrifuged at 1000 rpm for 15 min. An aliquot was mixed with a 5 mL solution of 20%Na2CO3, and the volume maintained at 10 mL using distilled water. After vortexing, theabsorbance of the mixture solution was determined at 750 nm.
Ascorbic acid (AsA). The method of [46] was used for the estimation of ascorbic acidcontent. Fresh wheat leaves were homogenized in 6% trichloroacetic acid (TCA) solutionand centrifuged. The supernatant was mixed with 2% dinitrophenyl hydrazine solution.After adding two drops of alcoholic thiourea the mixture was boiled then 5 mL of H2SO4(80% v/v) was added and the absorbance read using a spectrophotometer at 530 nm.
Total soluble sugars (TSS). Leaf soluble sugars were measured using the methodof [47]. The dried and powdered leaf samples were homogenized in a 10 mL solution of80% ethanol. An aliquot (0.1 mL) of the extract was taken after adding distilled water and4 mL of anthrone was added. Absorbance of the final solution was measured at 620 nm.
Enzymatic antioxidants SOD, POD, and CAT. Antioxidant activities of POD andCAT were determined by the method of [48], while the activities of SOD were determinedaccording to [49]. Leaf samples were extracted for these measurements in potassiumphosphate buffer.
Statistical analysis. The design of the experiment was completely randomized withthree replicates. The data on all attributes were subjected to a three-way analysis of variance(ANOVA) and treatment means were compared using least significant design test withStatistix 8.1 (Tallahassee, Florida, USA). The differences among means were calculated withthe least significance test level at a 5% probability level.
3. Results3.1. Growth Attributes
Drought stress (60% field capacity) was found to significantly (p ≤ 0.001) reduce theshoot length, root length and fresh and dry weights in both wheat cultivars comparedwith control conditions. Foliar application of both SA levels (3 and 6 mM) significantly(p ≤ 0.001) alleviated the deleterious effects of drought stress in all growth attributes in
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both wheat cultivars, as indicated in Figure 1. However, 6 mM SA foliar spray provedmore effective in improving the biomass, shoot and root length in both wheat cultivars.
Figure 1. Effect of foliar-application of salicylic acid on (A) shoot length, (B) root length, (C) shoot fresh weight, (D) scheme0., FSD-2008 (E) and SD-24 (F) Error bars above the means indicate standard errors (n = 3).
Drought stress decreased the shoot length of wheat by 9.65% and 17.14%, root lengthby 4.29% and 2.54%, shoot dry weight by 21% and 13%, and root dry weight by 15. 87%and 33.34% in S-24 and FSD-2008, respectively. Under drought stress, treatment with 6 mMSA increased the shoot length by 9.5% and 6.62%, root length by 7.32% and 3.13%, shootdry weight by 9.11% and 2.69% and root dry weight by 28.12% and 26.45% in FSD-2008
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and S-24, respectively, as compared with non-stress conditions. As indicated in Figure 1A,the shoot length of the Fsd-2008 cultivar significantly increased with foliar spray of SA.Likewise, root length, shoot fresh weight and shoot dry weight (Figure 1B–D) showedsimilar linear trends of increase after SA application in both cultivars. However, the growtheffects were more prominent in Fsd-2008 in comparison to S-24. The cv. Fsd-2008 cultivarhad greater improvement in shoot and root length, as well as biomass, compared withcv. S-24. Therefore, Fsd-2008 was highly drought tolerant and cv. S-24 was moderatelydrought tolerant regarding all growth parameters (Figure 1A–F; Table 1).
3.2. Photosynthetic Pigments
Results showed that drought stress considerably decreased the Chl a, Chl b andcarotenoid levels in both wheat cultivars. Furthermore, foliar spray with SA significantly(p ≤ 0.001) increased leaf photosynthetic pigments in both cultivars under control and stressconditions. Nevertheless, cultivar difference regarding the accumulation of photosyntheticpigments was also evident in that cv. Fsd-2008 accumulated more photosynthetic pigmentscompared cv. S-24 under both stress and nonstress conditions. Moreover, the interactionbetween drought stress, cultivars and SA application was significant (p ≤ 0.5), (p ≤ 0.01)for Chl b and carotenoids, respectively. Of the two cultivars, salicylic acid applicationimproved the Chl a content by 22.12% and 12.35%, Chl b content by 29% and 33.50% andcarotenoids by 14% and 20.92% under control and drought stress conditions, respectively,as compared with the no SA spray treatment (Figure 2A–C; Table 1).
3.3. Osmolyte Accumulation
A considerable increase in glycine betaine (GB) accumulation was recorded underdrought-stress in both wheat cultivars. The exogenous application of SA significantly(p ≤ 0.001) increased the accumulation of GB in both cultivars under stress and controlconditions. Cultivar difference was also apparent, as cv. S-24 accumulated more GBcompared cv. Fsd-2008. Of the two levels, SA at 6 mM resulted in a greater increasein the accumulation of GB as compared to 3 mM in both cultivars. (Figure 2F; Table 1).Proline and total soluble sugars were significantly (p ≤ 0.001) increased in both cultivarsunder drought stress conditions. However, under water-stress conditions, SA applicationsignificantly (p ≤ 0.001) increased the accumulation of proline and total soluble sugarsunder both stressed and nonstressed conditions. Between the two treatments, SA 6 mMresulted in a greater increase than 3 mM (Figure 2D,E; Table 1).
3.4. Oxidative Stress Attributes
A significant (p ≤ 0.001) increase in the activities of CAT and SOD in both the wheat cul-tivars was recorded when wheat plants were subjected to drought stress. Foliar-applicationof SA enhanced the CAT and SOD activities in both studied cultivars. The 6 mM SA levelwas more effective in increasing the activities of CAT and SOD in both wheat cultivars(Figure 3A,B; Table 1). Cultivar response regarding the activities of POD and SOD wasalso recorded. cv. Fsd-2008 had slightly higher activities of both these antioxidants, andwas highly drought-tolerant compared to cv. S-24, which was moderately drought-tolerant(Figure 3B,C; Table 1). Similar to the activities of CAT and SOD, the activity of POD wasalso significantly (p ≤ 0.01) increased in both wheat cultivars grown under drought stressconditions, while exogenous application of SA (3mM and 6 mM) further increased the PODactivity in both cultivars under drought stress and control conditions.
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Table 1. Mean square values from analysis of variance (ANOVA) of data for foliar-applied salicylic acid with respect to growth, photosynthetic pigments and oxidative defense systemsand osmolytes in wheat (Triticum aestivum L.) grown under water-stress conditions.
Source of Variance df SL RL SFW SDW RFW RDW Chl. a Chl. b Carotenoids MDA
Varieties (V) 1 87.11 *** 37.01 *** 73.67 *** 102.35 *** 9.45 *** 3.17 *** 7.40 *** 41.14 *** 0.08 *** 983.28 ***
Drought Stress (S) 1 35.60 *** 22.2 *** 44.67 *** 8.900 *** 0.51 *** 2..87 *** 13.04 *** 122.57 *** 0.04 *** 1787.99 ***
Treatments (T) 2 138.18 *** 23.38 *** 70.86 *** 126.97 *** 2.26 *** 1.48 *** 13.49 *** 28.89 *** 0.08 *** 1392.31 ***
V × S 1 4.27 * 0.47ns 1.17ns 1.03 * 0.07ns 0.27 * 0.38 * 5.26 *** 0.02 * 42.09 **
V × T 2 2.19ns 0.4ns 0.39ns 4.52 *** 0.03ns 0.01ns 0.03ns 2.49 *** 7.83ns 28.08 **
S ×T 2 3.03ns 0.32ns 2.24ns 18.13 *** 0.03ns 0.04ns 0.22ns 0.03ns 0.001 ** 347.64 ***
V × S × T 2 2.29ns 0.07ns 2.35ns 5.64 *** 0.01ns 0.24ns 0.01ns 0.22 * 8.13ns 24.72 *
Error 24 1.00 0.32 0.97 0.20 0.03 0.04 0.07 0.04 2.97 4.45
Source of Variance df H2O2 RMP Phenolics Ascorbic Acid Glycine betaine TSS Proline CAT SOD POD
Varieties (V) 1 0.36 ** 157.85ns 274.09ns 6.65 ** 82.75 *** 86.89 *** 4.59 *** 3374.62 *** 376.62 *** 160.94 ***
Drought Stress (S) 1 1.33 *** 157.85ns 4042.84 ** 75.23 *** 182.61 *** 393.56 *** 10.88 *** 2966.28 *** 47.60 *** 5.84 **
Treatments (T) 2 0.77 *** 290.40 ** 7836.06 ** 2.59 ** 86.32 *** 122.65 *** 38.28 *** 2180.47 *** 565.74 *** 155.29 ***
V × S 1 0.02ns 1.17ns 25.56ns 0.93ns 2.65ns 10.79 * 0.002ns 547.68 *** 0.01ns 19.70 ***
V × T 2 0.26 ** 0.94ns 4293.44 *** 0.24ns 1.11ns 3.19ns 1.56 *** 11.12ns 2.41ns 1.52ns
S ×T 2 1.26 *** 0.94ns 613.61ns 0.01ns 3.25ns 3.50ns 0.21 ** 29.92 * 8.12ns 10.33 ***
V × S × T 2 0.35 *** 0.49ns 57.12ns 0.80ns 0.66ns 1.31ns 2.87 *** 10.07ns 24.37 ** 18.51 **
Error 24 0.03 588.81 434.98ns 0.83 2.76 1.48 0.04 8.57 4.24 3.04
*, **, *** = significant at 0.05, 0.01 and 0.001 levels, respectively. ns = non-significant. Abbreviations: SL = shoot length; RL = root length; SFW = shoot fresh weight; RFW = root fresh weight; SDW = shoot dryweight; RDW = root dry weight; Chla = chlorophyll a, Chlb = chlorophyll b; Car = carotenoids; MDA = malondialdehyde; RMP = relative membrane permeability; H2O2 = hydrogen peroxide; total phenolics;ASC = ascorbic acid; GB = glycine betaine; TSS = total soluble sugars; Pro = proline; SOD = superoxide dismutase; POD = peroxidase; CAT = catalase.
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Figure 2. Effect of foliar-application of salicylic acid on (A) chlorophyll a, (B) chlorophyll b, (C) carotenoids, (D) proline,(E) total soluble sugar contents, (F) glycine betaine contents in two wheat cultivars under water-stress conditions. Meanswith the same letter (S) do not differ significantly at p ≤ 0.05. Error bars above the means indicate standard error (n = 3).
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Figure 3. Effect of foliar application of salicylic acid on (A) catalase, (B) superoxide dismutase, (C) peroxidase, (D) malondi-aldehyde, (E) hydrogen peroxide, (F) relative membrane permeability, (G) total phenolics, (H) ascorbic acid contents, in twowheat cultivars under water-stress conditions. Means with the same letter (S) do not differ significantly at p ≤ 0.05. Errorbars above the means indicate standard error (n = 3).
Compared to control plants, SA application increased the POD activity of S-24 andFSD-2008 by 60.07% and 47.12% under drought stress conditions, respectively. Cultivardifferences were also evident, cv. S-24 having less POD activity than cv. Fsd-2008, whichwas observed as drought tolerant (Figure 3; Table 1).
Leaf MDA and H2O2 levels increased considerably in plants of both cultivars un-der drought stress conditions. Exogenous application of SA (3 and 6 mM) significantly
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(p ≤ 0.001) decreased the MDA levels in both cultivars in stressed and control plants. SA6 mM was more effective in reducing the levels of both MDA and H2O2 in both cultivars.However, a nonsignificant increase was observed in relative membrane permeability (RMP)when plants were grown under drought stress. Exogenous application of SA significantly(p ≤ 0.01) inhibited the RMP level in stressed and nonstressed conditions (Figure 3D–F;Table 1).
Results showed that drought stress significantly (p ≤ 0.01) increased leaf phenoliccontents in both wheat cultivars under control and drought stress conditions. However,SA foliar-spray significantly (p ≤ 0.01) increased the phenolic content in both cultivarsunder drought stress and control conditions. Of the two treatments of SA, 6 mM wasmore effective in increasing phenolic contents in cv. S-24 (4.88%) and cv. Fsd-2008 (10.42%)(Figure 3G; Table 1). Under drought stress a considerable increase in leaf AsA content wasobserved in both cultivars. Exogenous application of salicylic acid at 6 mM significantly(p ≤ 0.01) further increased the accumulation of ascorbic acid in stressed and nonstressedplants. Cultivars difference was also apparent in that cv. Fsd-2008 accumulated highercontents of AsA compared to cv. S-24. Interaction between drought stress and salicylic acidspray was also significant (p ≤ 0.01) (Figure 3H; Table 1).
4. Discussion
The exposure of drought stress at 60% field capacity considerably reduced the shootand root attributes in the range of 9–17% for both wheat cultivars (Figure 1). Drought-induced reduction in growth attributes might be due to alteration in plant water relations,disturbance in photosynthetic pigments, photosynthetic activity and major damage at anoxidative level to macromolecules, as well as membrane deterioration and inhibition ofthe activity of photosynthetic enzymes [50]. In addition to this, drought stress reducescell division, cell expansion, the rate of photosynthesis, protein synthesis and nucleic acidmetabolism [51]. Previously, it was documented that crop plants use a well-developed rootsystem to avoid desiccation periods. In addition to this, root hydraulic properties and rootmorphology play vital roles in various responses to drought stress [52]. A healthy shootrequires an extensive root system, which may not available under drought conditions.Eventually shoot and root growth will be affected during periods of reduced water supplybecause the root system uses many photosynthetic end products for its growth [53].
Foliar-spray with SA improved all growth attributes, such as shoot and root length,as well as fresh and dry weights in both the wheat cultivars grown under drought stressand control conditions (Figure 1). In the two wheat cultivars, 6 mM SA-spray enhancedthe shoot length by 11.35% and 20.35% and root length by 7.32% and 3.13% under controland drought stress conditions, respectively. Foliar spray of salicylic acid is potentiallyeffective to exert a suppressive or simulative influence on growth aspects through its directinterference with key enzymatic activities responsible for biosynthesis and metabolism ofgrowth-promoting substances [30,54]. In this context, many researchers found that salicylicacid improved growth in maize [55,56] and wheat [57]. Similarly, the growth-promotingresponse of salicylic acid in onion was reported earlier by [30,56]. Our results relate to thestudy of [58] in lemongrass in which the dry weights of shoots and roots were increased byfoliar spray of salicylic acid under drought stress.
The present investigation revealed that Chl a Chl b, as well as carotenoid contents,decreased in both wheat cultivars when subjected to drought stress (Figure 2). The drought-induced decrease in chlorophyll pigments might have been due to inhibition in vitalprocesses of photosynthesis, such as chlorophyll biosynthesis, which ultimately decreasedchlorophyll contents [59]. Reduction in chlorophyll content could, ultimately, decreasephotosynthetic rate [60]. These results relate to the finding of [61] in wheat and [62]in cucumber in which chlorophyll contents decreased under drought stress conditions.Nevertheless, several researchers found that decrease in chlorophyll content was due toover-production of reactive oxygen species (ROS), degradation in nutritional balance andinactivation enzyme activities, as well as a decline in cellular water contents [63–65].
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Our results indicate that foliar spray of SA increased chlorophyll pigments in bothwheat cultivars under stress and control conditions (Figure 2). The beneficial effects ofSA with respect to the deleterious effects of drought on chlorophyll pigments could beattributed to its stimulatory effects on Rubisco activity and, ultimately, to increased photo-synthetic rate. Other positive effects of SA could be linked to induction of the synthesisof protein kinases, which have key roles such in the regulation of cell division and atvarious cell developmental stages [27,66]. Other reports showed that SA increased pho-tosynthetic rate and stomatal conductance under stressful environmental conditions [67].Moreover, [31] found that SA improved biosynthesis and enhanced photosynthetic ratein corn and soybean. Compared to controls, an SA application of 6 mM SA increased theChl a content by 22.12% and 12.35% and Chl. b content by 29.53% and 35.5% in S-24 andFSD-2008, respectively.
In the present investigation, the imposition of drought stress in wheat plants in-creased osmolytes such as proline, glycine betaine and total soluble sugars in both cultivars(Figure 2). In this respect, to deal with adverse effects of drought stress, plants carried outthe process of osmotic adjustment by the synthesis of osmolytes such as GB, proline, andtotal soluble sugars. Proline and GB are involved in cellular osmotic regulation. Proline,being a secondary metabolite, is an antioxidant as well as an osmoprotectant [68]. As anantioxidant it is has a protective role in the membranes of organelles, and as an osmolyte itplays a vital role in regulating cellular water relations under drought stress conditions [69].It was documented by Habib et al., [68] that during osmotic stress conditions prolineacts as water substituent that stabilizes intercellular structures via hydrophobic interac-tions and hydrogen bonding, eventually protecting the membrane from dehydration. Itwas also documented that increased concentration of glycine betaine also plays a roleby stimulating novel stress-related genes, detoxifying ROS, as well as a protective rolein the photosynthetic machinery and maintenance of protein structures, by acting as amolecular chaperone under stressful conditions [69]. Our results are inconsistent with theinvestigation of Damghan et al. [70] and Zhang et al. [71] who found that proline acts asan osmolyte rather than in protecting enzymes and other macromolecules, providing aprotective mechanism against low water potential conditions via osmotic regulation andacting as an electron acceptor, preventing photosystem injuries by scavenging ROS.
In the present study, foliar-application of SA was found to increase the accumulationof osmolytes under drought stress and control conditions. Between the two levels of SA,4 mM and 6 mM, 6 mM further increased the osmolyte proline in both wheat cultivarscompared to controls (Figure 2). The exogenous application of SA further enhanced prolineaccumulation in water-stressed plants. A similar study by Umebese et al. [72] reportedthat SA-application protects amaranthus plants by enhancing proline accumulation. Foliarapplication of salicylic acid increased the accumulation of total soluble sugars in onion [73].Similarly, it was reported that SA provided a protective role against drought stress intomato and amaranthus [72]. Our results are consistent with the work of Idrees et al. [66]with lemongrass, of Bakry et al., [74] with linseed, and of [75] in with wheat, who reportedthat SA application increased the proline accumulation under drought stress, and wasinvolved in protective mechanisms mitigating against stress.
The present investigation revealed that the imposition of drought stress increased thecontents of MDA and H2O2 in both the wheat cultivars (Figure 3). MDA accumulationwas measured in the form of lipid peroxidation. Over-production of MDA is an indica-tor of oxidative damage [76]. The oxidation of membrane lipids is an indication of theformation of free-radicals producing oxidative stress [77]. Similarly, several researchersshowed that increased level of MDA and H2O2 triggered ROS-induced oxidative dam-age under drought stress [9,59,76]. Similarly, drought-induced oxidative stress enhancedthe accumulation of MDA, and H2O2 has been found in wheat [78], cucumber [79] andcanola [61] A nonsignificant increase in the level of relative membrane permeability wasobserved under wheat cultivars under drought stress conditions (Figure 3). Drought causesdeleterious effects such as inactivation of enzyme activities, damage to cell membranes,
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ion-leakage and reduced osmotic regulation due to the higher concentration of ROS in-side cells [80–82]. Our results indicated that activities of the antioxidant enzymes, SOD,POD and CAT increased when wheat plants were subjected to drought stress conditions.(Figure 3). The formation of the O2-radical is a basic reason for oxidative injury, and manysystems are used by plants to mitigate its effects in drought tolerance [83]. Enhancedactivity of SOD related to dismutation/modulation of O2 into H2O2 maintains the cellularoxidation state at a normal level and is considered as the protective role of SOD againstoxidative stress [84]. In this regard, O2 generation inactivates SOD activity [85]. Mani-vannan et al. [86] found that enhanced SOD activity is a trend towards drought tolerance.Noctor et al., [87] found that during drought stress conditions increased SOD activity is anindicator of drought tolerance in tomato. CAT also has the potential to mitigate oxidativestress [88]. The increased concentration of CAT and SOD converts toxic O2 to H2O2 andultimately into water and molecular oxygen, reversing damage under adverse conditions,so both these play a role in scavenging ROS at the cellular level [89]. Similar results havebeen reported by several researchers who found that enhanced CAT and SOD becomeoxidative scavengers to detoxify radicals under stressful environments [90,91]. Oxidativedefense systems comprised of the enzymes CAT, POD, and SOD, and the nonenzymaticascorbic acid, phenolics, flavonoids, and proline, detoxify stress-induced ROS accumu-lation, [76,92,93]. Drought-induced increases in the activities of enzymatic antioxidantssuch as CAT, POD and SOD were reported earlier by Wang et al., [94] in grapes understress conditions. Ashraf et al., [12] and Akram et al., [61] reported that SOD plays anessential role in detoxification, while POD and CAT are involved in countering severallatent oxidants to recover stress tolerance. The upregulation of antioxidants in stressedconditions is an indicator of stress tolerance [95,96]. Canola and radish cultivars werestudied under stress conditions and found to be relatively tolerant based on enhancedactivities of these antioxidants [61,97].
Foliar application of SA significantly reduced H2O2 and MDA levels, and stabilizedmembrane stability in wheat seedling compared to control plants, which indicated that ROSproduction during water-stress conditions was effectively overcome by the SA-foliage sprayreducing oxidative damage (Figure 3). Between the two levels (3 mM and 6 mM), 6 mMwas more effective in reducing water-stress damage in both cultivars. Foliar-application ofSA further enhanced the activities of antioxidant enzymes such as SOD, POD, and CATunder stressed and nonstressed conditions, which indicated lower accumulation of MDAand H2O2 and lesser electrolyte leakage, thereby providing membrane stability (Figure 3).It was reported by Fobert and Despres [98] that SA application plays a key role in theactivation of antioxidants such as SOD, POD and CAT to scavenge ROS via activation of adefensive system triggering redox changes in signal transduction pathways. Previously,it was documented that foliar spray of SA upregulated antioxidant activities under stressconditions [99]. It has been shown that SA-application causes an increase in the activity ofantioxidants in various stress situations to remove ROS [91].
In the present study, nonenzymatic antioxidants, such as total phenolics and ascorbicacid, were increased under drought stress (Figure 3). These nonenzymatic antioxidantsdetoxify ROS during oxidative stress [22]. Similarly, it was found that antioxidants in-creased under drought stress in the work of Ahmad et al., [28] in canola and Shafiq et al. [22]in corn. However, in contrast, a high level was found in carrot [62], while AsC level in-creased in canola [22]. Furthermore, phenolics and ascorbic acids help in the regulation ofmetabolism and in reducing oxidative-damage [13]. SA-application increased phenolic andascorbic acid contents [53,73,74] and enhanced the level of all antioxidant enzymes (enzy-matic and nonenzymatic) resulting in detoxification of oxidative stress in wheat seedlings(Figure 3). In conclusion, foliar-spray improved the resistance of plants against droughtstress by upregulating the antioxidant defense system of all the antioxidant enzymes andreducing the oxidative damage caused by reactive oxygen species (ROS) [5,66,100]. Furtherresearch will be focused on the influence of a synthetic form of salicylic acid on Triticumaestivum under field conditions [101].
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5. Conclusions
It can be concluded that foliar spray with salicylic acid was very effective in mitigatingthe adverse effects of water-stress in wheat. Foliar-application, particularly at 6 mM,significantly enhanced growth attributes which were associated with increased biosynthesisof photosynthetic pigments, as well as cellular osmotic adjustment by the accumulationof osmolytes. Induction of water stress tolerance in wheat plants after foliar spray withSA was also associated with the antioxidative defense mechanism through increases in theactivities of antioxidant enzymes (SOD, POD, and CAT) and accumulation of nonenzymaticantioxidants, which actively reduced membrane lipid peroxidation by lowering the levelof MDA and H2O2 in both cultivars. Future research may be focused on confirming theobtained results under uncontrolled (field) conditions.
Author Contributions: Conceptualized, executed the work, wrote the first draft of the paper, A.P.;analyzed the data, M.A.A.; Data analysis and partly helped in paper writing, I.H., Q.M. and A.D.;helped to develop graphs and presentation of paper, S.P.; helped to carry out experiment, R.R.;helped in paper refining and checking of paper, S.H.; helped to do English editing and final revision,A.H., E.F.A.A., and A.-B.F.A.-A.; statistical analysis, A.A.A. All authors have read and agreed to thepublished version of the manuscript.
Funding: The facilities provided at GCUF are acknowledged. The authors would like to extend theirsincere appreciation to the Deanship of Scientific Research at King Saud University for funding thisresearch group NO (RG-1435-014).
Data Availability Statement: Available on request from corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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